Searching for pain-preferential neural activity is essential for understanding and managing pain. Here, we investigated the preferential role of thalamocortical neural dynamics in encoding pain using human neuroimaging and rat electrophysiology across three studies. In study 1, we found that painful stimuli preferentially activated the medial-dorsal (MD) thalamic nucleus and its functional connectivity with the dorsal anterior cingulate cortex (dACC) and insula in two human functional magnetic resonance imaging (fMRI) datasets (n = 399 and n = 25). In study 2, human fMRI and electroencephalography fusion analyses (n = 220) revealed that pain-preferential MD responses were identified 89-295 ms after painful stimuli. In study 3, rat electrophysiology further showed that painful stimuli preferentially activated MD neurons and MD-ACC connectivity. These converging cross-species findings provided evidence for pain-preferential thalamocortical neural dynamics, which could guide future pain evaluation and management strategies.